Effect of chitosan coated chemogenic silver nanoparticles coated syringes against biofilm of clinical isolate of Staphylococcus aureus

International Journal of Advancements in Research & Technology, Volume 3, Issue 6, June-2014 35 ISSN 2278-7763

Copyright © 2014 SciResPub. IJOART

Effect of chitosan coated chemogenic silver nanoparticles coated syringes against biofilm of clinical isolate of Staphylococcus aureus S.Karthick Raja Namasivayam, Pawan Kumar, S.Kiran Nivedh, A.N.Nishanth, EAllen Roy Department of Biotechnology, Sathyabama University, Chennai 119, Tamil Nadu, India *Corresponding author e-mail address [email protected] ABSTRACT

Biofilm represents the most prevalent type of virulent factor of most of the pathogenic microorganism

and involved in crucial development of clinical infection and exhibit resistance to antimicrobial agents.Now the

biofilm is considered as major target for the pharmacological development of drugs. A biofilm serves to

promote bacterial persistence by resisting antibiotic treatment and host immune responses. Antibiotics are

rendered ineffective when biofilms form due to their relative impermeability, the variable physiological status of

microorganisms, subpopulations of persistent strains, and variations of phenotypes present .Metal

nanotechnology chemistry has the potential to prevent the formation of these life-threatening biofilms on life

supporting devices.In the present study, anti biofilm effect of silver nanoparticles coated syringes against

clinical isolate of Staphylococcus aureus was studied. Chitosan stabilized silver nanoparticles synthesized by

chemical reduction method and the synthesized particles were coated on the surface by ultrasonication. Coated

syringes were characterized by scanning electron microscopy (SEM) which reveals complete dispersion of the

nanoparticles on the fibre surface and the size, shape of the particles shows uniform spherical particles with the

size of 60-70 nm. Distinct effect of biofilm inhibition was recorded in the nanoparticles coated syringes and

maximum inhibition was observed during 72 hour of incubation. Biochemical composition of biofilm matrix

mainly total carbohydrates and total protein was highly reduced. The present study would suggests the

development of anti microbial coated medical devices against pathogenic microorganism.

Keywords. Biofilm, Staphylococcus aureus,Silver nanoparticles,Chitosan

1.INTRODUCTION

Nanobiotechnology, the convergence of nanotechnology and biotechnology and in particular

its applications in the medical sector are considered as one of the most promising and most advanced

areas of nano technology[1]. The application of nanotechnology in the field of healthcare has come

under great attention in recent times. There are many treatments today that take a lot of time and are

also very expensive. Using nanotechnology, quicker and much cheaper treatments can be developed.

By performing further research on this technology, cures can be found for diseases that have no cure

today. The application of such a technology can be used for the inhibition of biofilm formation on the

surgical and medical devices which are of higher threat in the process of treatments. Bacteria are able

to grow adhered to almost any surface, forming architecturally complex communities termed biofilms

[2,3]. Microbial biofilms develop when microorganisms irreversibly adhere to a submerged surface

and produce extracellular polymers that facilitate adhesion and provide a structural matrix. This

surface may be inert, nonliving material or living tissue. Biofilm-associated microorganisms behave

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differently from freely suspended organisms with respect to growth rates and ability to resist

antimicrobial treatments and therefore pose a public health problem [4, 5]. Due to increasing tolerance

of the biofilm community to antibiotics, biocides and mechanical stress, it has become just as difficult

to completely eradicate mature biofilms as it is to completely avoid the presence of planktonic cells,

the origin of the biofilm in the water. Common treatments to prevent or remove biofouling include

using disinfection, minimizing nutrients in the feed or altering surface materials to prevent bacterial

attachment, or clean-in-place (CIP) to remove mature biofilm by chemical or mechanical shear.

Several studies have examined the effect of various types of antimicrobial treatment in controlling

biofilm formation on medical devices[6, 7, 8]. The vast majority of the chemical agents currently

available for biofilm control are broad-spectrum non-specific micro biocide agents [9]. Chloro

hexidine, triclosan, and essential oils (e.g., Listerine) are the most commonly used and clinically tested

antimicrobials [10]. Biofilm-control strategies based on disruption of EPS formation on the surface

could be an effective alternative (or adjunctive) approach [11]. In order to control biofilm formation

on medical devices and all costs associated, a large number of new strategies and approaches have

been developed in the last few years, including: antimicrobial locks (in the case of catheters) [12];

surface modification of biomaterials with antimicrobial coatings [13]; the use of quorum sensing (QS)

inhibitors [14], antimicrobial peptides as a new class of antibiotics [15]; enzymes that dissolve

biofilms [16], nitric oxide [17], electrical [18] or ultrasound [19] enhancement of antimicrobial

activity, or even the application of light activated antimicrobial agents [20]. Nevertheless, nanoscale

materials have recently appeared as one of the most promising strategies to control biofilm infections

related to indwelling medical devices, especially due to their high surface area to volume ratio and

unique chemical and physical properties [21]. A nanomaterial has a diameter ranging from1 and 100

nm, and they can be made from different materials, like copper, zinc, titanium, magnesium, gold,

alginate and silver. The use of silver nanoparticles (NPs) is now considered as one of the most

promising strategies to combat biofilm infections related to indwelling medical devices [22]. Drug

delivery nano carriers systems, such as liposomes [23] and polymer-based [24] carriers have also

arisen as appealing methods with a great potential in the treatment of biofilm infections, due to several

factors especially good biocompatibility and ample range and extent of drugs that they can carry.

Another important factor is the protection provided by the encapsulation of the drug in the biological

milieu, decreasing toxicity and allowing the drug to reach the specific site [25]. Chitosan is another

natural polymer has been reported as a polymer-based protective agent to stabilize the metal

nanoparticles[26].Because of the biocompatibility,biodegradability, nontoxicity and adsorption

properties of chitosan, it was used as a stabilizing agent to prepare Ag, Au and Pt nanoparticles.

These chitosan- protected nanoparticles can be easily integrated into systems relevant for

pharmaceutical, biomedical, and biosensor applications. Therefore, it has attracted considerable

interest due to its medicinal properties, such as antifungal, antibacterial, antiprotozoal, anticancer,

antiplaque, antitartar, hemostatic, wound healing and potentiates anti-inflammatory response, inhibits

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the growth of cariogenic bacteria, immunopotentiation, antihypertensive, serum cholesterol lowering,

immune enhancer, increases salivary secretion (anti-xerostomial) and helps in the formation of bone

substitute materials[27].The present study is aimed to evaluate anti biofilm effect of biocompatible

polymer stabilized metallic nanoparticles coated syringes against clinical isolate of Staaph. aureus

under in vitro condition.

2.MATERIALS AND METHODS

Coating of metallic nanoparticle on syringes

Syringes were obtained from Solaguard (Chennai,Tamil Nadu,India). The outer transparent portion of

syringes were cut into 4 pieces and transferred to beaker containing 0.1 molar AgNO3 and 0.1molar tri

sodium citrate placed in ultrasonicator. Freshly prepared 0.1 molar sodium borohydride was added

drop by drop till reaction mixture turned into brown. The preparation was left in ultrasonicator for 2

hours to facilitate complete dispersion of nanoparticle on surface. The coating of nanoparticles on

syringe was primarily confirmed by colour change of cut pieces of syringe into brown colour. The

pieces were dried at 40oC overnight to remove excess moisture. The dried pieces were kept in sterile

petriplate for further study. Chitosan coated nanoparticle was also coated by chemical reduction

method of respective metal precursor with reducing agent and 0.1 molar chitosan as a stabilizer agent.

Chitosan was obtained from SRL laboratory and deacetylation process was done and degree of

deacetylation was determined using Viscometric method. The pre-treated chitosan as described earlier

was dissolved in 1% w/v acetic acid (1 mL of acetic acid in 100 mL of distilled water) and suspension

was transferred to a beaker containing respective metal precursor and a reducer. The homogenous

slurry thus obtained was coated with cut pieces. Before coating, the suspension was characterized by

scanning electron microscopy (SEM equipped with energy dispersive x ray atomic spectroscopy

(EDAX), the mixtureThe coated cut pieces thus obtained were dried at 40oC as described earlier.

Biofilm inhibition assay

The metallic nanoparticles coated syringes kept in sterile Petri plates were inoculated with 5 mL of

S.aureus culture. The plates were allowed for incubation at 37oC for 72 hours. After incubation period

the treated syringes were stained with 0.1% w/v of crystal violet solution for 15 minutes at room

temperature. After staining the syringe pieces were washed with phosphate buffered saline (PBS)

solution to remove free planktonic cell. Further washing was carried out with 95% of ethanol for 3

times at room temperature and the washed solution was collected and absorbance was measured

spectrophotometrically at 540nm.

The percentage of biofilm inhibition was calculated by following formula:

Biofilm inhibition (%) = OD in control – OD in treatment × 100

OD in control

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Biofilm Kinetics

Biofilm kinetics was done to study the inhibition percentage of nanoparticles coated syringes

against biofilm of Staphylococcus aureus with respect to time. Fresh syringe was taken and dispersed

in metallic nanoparticles - AgNp, AgNp-CS. The syringe was kept in beaker containing AgNp and

kept for sonication in water bath sonicator for 3 hours likewise performed for AgNp-CS and kept them

in water bath sonicator for 3 hours. Once coating was done, the Petri plates were kept in dry air oven

at 46oC for 2 hours. After drying, inoculation was done by spraying 5 mL of Staphylococcus aureus on

different nanoparticle coated syringes and kept for incubation for different time interval. Fresh syringe

was taken as control in another petri plate. After 12 hours of incubation, Ag coated syringe was cut

into 1st part with surgical blade and the remaining part was kept for further incubation. Similarly

AgNp-CS coated syringe was cut and the remaining portion was kept for incubation. Incubated coated

syringes and control syringes were dipped in 2 mL of 0.1% w/v of crystal violet in sterile boiling test

tubes each, shaken properly and kept for incubation at room temperature for 15 minutes. After the

incubation period crystal violet was removed with sterile micro tip then the syringes were washed with

2 mL of sterile phosphate buffered solution twice. It was aspirated and PBS was discarded, 5 mL of

ethanol was added to each tube and was kept on ultrasonicator for 15 minutes. Elutants were measured

at 540nm spectrophotometrically and reading was kept for tabulation.

Evaluation of effect of nanoparticles on biochemical composition of biofilm matrix

Biochemical composition of biofilm matrix mainly total carbohydrate and total protein was

carried out. The control syringe pieces and respective nanoparticle coated syringe pieces (3 in each

treatment) was transferred to test tube each containing 5 mL of culture containing S.aureus prepared

overnight. Test tubes containing pieces and culture were kept for incubation at 37oC for 3 days for

allowing formation of biofilm on the syringes

After incubation period, the inoculated pieces were transferred to screw cap vials containing 5

mL of 0.9% NaCl. The bottles were sonicated for 10 minute in an ultrasonicator water bath and

vortexed vigorously for 1 minute to disturb biofilm. Cell suspensions were then folded and centrifuged

at 10000 rpm at 4oC for 10 minutes.The collected suspension was used as source for studying

biochemical composition in terms of total protein determined by Lowry et al and total carbohydrate by

Anthrone method.

3.RESULT AND DISCUSSION

Chitosan stabilized silver nanoparticles were synthesized by chemical reduction of metal salt

precursor with nontoxic and biocompatible polymer chitosan which primarily confirmed by

FTIR.SEM and EDAX. When the FTIR spectrum of free and stabilized nanoparticles were

compared, it was found that almost the all the absorbed peaks were modified upon coating with

chitosan. FTIR spectra of chitosan coated silver nanoparticles are presented in (Figure 1 a,b).The IR

spectra of the chitosan capped Nano silver shows prominent peaks at ≅ 3788 cm-1, 3427.4005 cm-1

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corresponding to O – H stretching, strong polymerization, at 2928.1733 cm-1 for aliphatic C – H

stretching. Peaks at approximately 2369.0387 cm-1,2345.3187 cm-1 represent N – H stretching

vibration. Peaks at 1637.5845 cm-1 and 1389.4649 cm-1 represent N –H bending and a peak at

1026.6957 represents C – N vibration in aliphatic compounds. A is also observed at 617.7611 cm-1

showing the presence of inorganic metal ions (silver ions). SEM analyzer built in with and EDAX

analyzer allows a quantitative deduction on localization of elements in the nano specimens Scanning

electron microscopy (SEM) study of chitosan stabilized silver nanoparticles reveals. the uniform

spherical smooth morphology. within the size range of 101.78 nanometers and electron dense thin

chitosan coating shell of diameter 3-5 nanometers(Figure2 a) Such size distribution analysis primarily

confirms that the particles are well dispersed and less aggregated The EDAX images illustrated the

presence of large amounts of C, O, N (Figure 2 b).

Coating of respective nanoparticles on syringes by ultrasonicator was primarily confirmed by

fine dispersion of particles on the surface which can be easily visualized. Surface topography with

SEM clearly reveals uniform spherical particles with nano range embedded on the syringe surface

(Fig. 3.) SEM Micrograph reveals complete disturbance of biofilm, less aggregates, weakened cell

mass was observed (Figure 3a). Biofilm inhibition study clearly reveals all the nanoparticles (both free

and coated) inhibited biofilm in significant manner (P.0.05). Maximum inhibition of 80.4% was

recorded in AgNp-CS treatment. followed by. 72.8% was reported from free Ag and (Table 1).

Biofilm kinetics Biofilm kinetics study clearly reveals all the tested nanoparticles inhibited biofilm with

respect to different time interval ranging from 12, 24, 36, 48, 60, 72 hours, but distinct effect was

observed in chitosan coated nanoparticle and linear increase in inhibitory effect was inferred during

late inhibition period.

In the case of free AgNps the biofilm inhibition at respective time period was found to be 9.8,

11.7, 15.9, 21.05, 34.3, 41.6 % (Table .2.). Improved inhibitory activity was reported from CS coated

AgNp as 11.2, 14.6, 19.3, 26.1, 40.7, 49.8 % during 12, 24, 36, 48, 60, 72 hours respectively (Table

3.)

Effect of Nanoparticles on biochemical composition of biofilm matrix of S.aureus

All the tested nanoparticles (both free and coated) reduced biochemical composition mainly

total protein and total carbohydrate of biofilm matrix (Table 4). Maximum reduction of protein was

recorded in chitosan coated syringes (12 mg/mL) followed by AgNp-CS (15 mg/mL). free Ag

nanoparticle recorded was (17 mg/mL).Similar effects on carbohydrate content was also reported

from CS and AgNp-CS nanoparticles which recorded least total carbohydrate content (14 mg/mL)

each followed by AgNp (16 mg/mL).

The developments of nanoparticles with antimicrobial properties have recently

received growing interest from both academic and industrial sectors due to the increasing

resistance of pathogenic microorganisms to the diverse conventional chemotherapeutics.The

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present study demonstrated that silver nanoparticles synthesized by chemical reduction

method and stabilized with biocompatible polymer chitosan coated on the syringes showed

distinct anti biofilm effect against Staphylococcus aureus can be used in to prevent or to

minimize bacterial infections and will lead to new generation of development of

antimicrobial agents to prevent pathogens infection

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Table 1 Biofilm inhibition (%) of S.aureus with metallic nanoparticles

Serial No. Treatment Biofilm inhibition (%)

1 AgNp 72.8

2 AgNp-CS 80.4

Table .2: Biofilm inhibition (%) of S.aureus with Silver Nanoparticles (AgNps)

Table 3. Biofilm inhibition (%) of S.aureus with Chitosan coated Silver Nanoparticles (AgNp-

CS)

Serial No. Time-Interval (hrs.) Biofilm inhibition (%)

1 12 11.2

2 24 14.6

Serial No. Time-Interval (hrs.) Biofilm inhibition (%)

1 12 9.8

2 24 11.7

3 36 15.9

4 48 21.05

5 60 34.3

6 72 41.6

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3 36 19.3

4 48 26.1

5 60 40.7

6 72 49.8

Table 4. Effect of Free and Chitosan coated metallic nanoparticles coated syringes against

biofilm matrix biochemical composition of S.aureus.

Treatment

Total Carbohydrate

(mg/mL) Total Protein (mg/mL)

AgNp 16 17

AgNp-CS 14 15

Figure 3.SEM image of nanoparticles coated syringe

Figure 3 a.Biofilm of Staph.aureus on un coated syringe

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Figure 3b.SEM image of nanoparticles coated syringe showed disturbed biofilm

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Documents

Effect of chitosan coated chemogenic silver nanoparticles coated syringes against biofilm of clinical isolate of Staphylococcus aureus